Cardiomyocyte apoptosis is now accepted to be a major contributor to cell death induced by ischemia/ reperfusion (I/R) as well as to the development of dilated cardiomyopathy and heart failure. In the heart, as in isolated cardiomyocytes, the serine threonine kinase Akt protects against I/R injury and apoptosis induced by various cell stresses. Akt is activated through growth factor stimulation of PI3 kinase, an enzyme that has been well studied in the heart. The other enzymes that regulate Akt phosphorylation have not been explored in cardiomyocytes although these are likely drug targets for cardioprotection. These include the immediate upstream kinase PDK-1, and phosphatases responsible for Akt dephosphorylation amongst which is the recently discovered phosphatase, PHLPP. In Aim # 1 we address the regulation and role of PHLPP and the requirement for PDK-1 in Akt phosphorylation and activity in cardiomyocytes, using PHLPP and PDK-1 knockout mice. We further examine how loss of these enzymes affects ischemic reperfusion damage in vivo and the development of apoptosis, mitochondria! disruption and autophagy in vitro. A great many studies of Akt have used activated membrane targeted forms of the enzyme. It has also become increasingly clear, however, that Akt has actions at multiple sites including the plasma membrane, cytosol, nucleus and mitochondria. The cellular location and dynamic control of Akt by its regulatory enzymes can lead to a range of spatiotemporal signals which will in turn dictate the nature of the downstream signaling events. Thus whether Akt activation regulates cytosolic enzymes, nuclear gene expression or mitochondrial integrity may depend upon its activation kinetics and location. Accordingly in Aim # 2 we propose to examine the temporal and spatial aspects of Akt regulation using a FRET based Akt activity reporter (BKAR) and other BKARS targeted to specific cellular locations. The possibility that mTOR acts not only as a downstream target of Akt but as a feedback regulator of Akt dephosphorylation will be examined in Aim # 3, testing the hypothesis that rapamycin affects Akt dephosphorylation through PHLPP or regulates kinase mediated Akt phosphorylation. The possibility that rapamycin affects the cells decisions to undergo autophagy through regulation of Akt will also be examined. We recently demonstrated that Akt associates with mitochondria in cardiomyocytes, and in particular with components of the mitochondrial permeability transition (PT) pore including hexokinase (HKII). In Aim # 4 the regulation of the HKII expression, its association with mitochondria, and its phosphorylation by Akt are examined. The role of HKII in mediating the cardioprotective effects of Akt on the mitochondrial permeability transition pore is tested. Elucidation of novel enzymatic pathways regulating Akt phosphorylation, and information on the compartmentalized effects of this enzyme, will allow design of therapeutic interventions that can selectively target components of this pleiotropic signaling pathway.